Chromatin profiling identifies chondrocyte-specific Sox9 enhancers important for skeletal development
Sachi Ichiyama-Kobayashi, Kenji Hata, Kanta Wakamori, Yoshifumi Takahata, Tomohiko Murakami, Hitomi Yamanaka, Hiroshi Takano, Ryoji Yao, Narikazu Uzawa, Riko Nishimura
Sachi Ichiyama-Kobayashi, Kenji Hata, Kanta Wakamori, Yoshifumi Takahata, Tomohiko Murakami, Hitomi Yamanaka, Hiroshi Takano, Ryoji Yao, Narikazu Uzawa, Riko Nishimura
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Research Article Bone biology

Chromatin profiling identifies chondrocyte-specific Sox9 enhancers important for skeletal development

Abstract

The transcription factor SRY-related HMG box 9 (Sox9) is essential for chondrogenesis. Mutations in and around SOX9 cause campomelic dysplasia (CD) characterized by skeletal malformations. Although the function of Sox9 in this context is well studied, the mechanisms that regulate Sox9 expression in chondrocytes remain to be elucidated. Here, we have used genome-wide profiling to identify 2 Sox9 enhancers located in a proximal breakpoint cluster responsible for CD. Enhancer activity of E308 (located 308 kb 5′ upstream) and E160 (located 160 kb 5′ upstream) correlated with Sox9 expression levels, and both enhancers showed a synergistic effect in vitro. While single deletions in mice had no apparent effect, simultaneous deletion of both E308 and E160 caused a dwarf phenotype, concomitant with a reduction of Sox9 expression in chondrocytes. Moreover, bone morphogenetic protein 2–dependent chondrocyte differentiation of limb bud mesenchymal cells was severely attenuated in E308/E160 deletion mice. Finally, we found that an open chromatin region upstream of the Sox9 gene was reorganized in the E308/E160 deletion mice to partially compensate for the loss of E308 and E160. In conclusion, our findings reveal a mechanism of Sox9 gene regulation in chondrocytes that might aid in our understanding of the pathophysiology of skeletal disorders.

Authors

Sachi Ichiyama-Kobayashi, Kenji Hata, Kanta Wakamori, Yoshifumi Takahata, Tomohiko Murakami, Hitomi Yamanaka, Hiroshi Takano, Ryoji Yao, Narikazu Uzawa, Riko Nishimura

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Figure 3

Correlation of identified enhancer activity with Sox9 expression.

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Correlation of identified enhancer activity with Sox9 expression. (A) Co...
(A) Comparison of Sox9 mRNA levels in primary chondrocytes (1°Cho), 2nd passage chondrocytes (2nd Cho), and dermal fibroblasts (DF) using RT-qPCR. Data are shown as the mean ± SD (n = 3, biologically independent samples). **P < 0.01; 1-way ANOVA followed by Tukey’s multiple-comparison test. (B) Comparison of Sox9 protein between 1°Cho, 2nd Cho, and DF by Western blot. (C) Luciferase reporter plasmids that include the E160 or E308 enhancer and a Sox9 minimal promoter were transfected into 1°Cho, 2nd Cho, and DF cells. Luciferase activity was measured 48 hours after transfection. Data are shown as the mean ± SD (n = 4, biologically independent samples). **P < 0.01, 1-way ANOVA followed by Tukey’s multiple-comparison test. (D) Comparison of Sox9 mRNA in different cell lines by RT-qPCR. Data are shown as the mean ± SD (n = 3, biologically independent samples). (E) Comparison of Sox9 protein in different cell lines by Western blot. (F) Luciferase reporter plasmids as in C were transfected into cell lines. Luciferase activity was measured 48 hours after transfection. Data are shown as the mean ± SD (n = 4, biologically independent samples). (G) Chondrocyte differentiation of ATDC5 cells. ATDC5 cells were cultured with or without insulin, transferrin, and selenium (ITS) for 21 days and then stained with Alcian blue. (H) Total RNA isolated from ATDC5 cells was analyzed by RT-qPCR for Sox9 gene expression. Data are shown as the mean ± SD (n = 3, biologically independent samples). **P < 0.01 (vs. Control). (I) ChIP-qPCR analysis of E308 and E160 in ATDC5 cells treated with or without ITS. Sonicated chromatin isolated from ATDC5 cells was immunoprecipitated with anti-H3K27ac antibody and quantified using specific primers for E308 and E160. Data are shown as the mean ± SD (n = 3, biologically independent samples). **P < 0.01 (vs. Control); unpaired Student’s t test.